Sensor arrays with nucleophilic indicators

ABSTRACT

Sensors arrays that include indicators arranged in a pattern on a substrate. The indicators include nucleophilic indicators. In some examples, the sensor arrays with multiple nucleophilic indicators provide superior detection and identification of microorganisms, cancer biomarkers, formaldehyde, organophosphates, and aldehydes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/897,403, filed Feb. 15, 2018 which claims priority to and the benefitof U.S. Provisional Patent Application No. 62/459,795, filed Feb. 16,2017 and entitled “Sensor Arrays with Nucleophilic Indicators,” thecontents of which are hereby incorporated by reference in their entiretyas if fully set forth herein.

FIELD

This disclosure relates to artificial noses for the detection ofanalytes.

BACKGROUND

Array-based sensors mimic the mammalian gustatory and olfactory systemsto detect and characterize fluidic samples by measuring a uniquecomposite response for each analyte rather than an analyte specificsensor. Such cross-responsive sensor arrays have been implemented bothas an artificial nose technology for the detection of vapors and gases,and as electronic tongue technology for the detection of liquidanalytes. Examples include, but not limited to: colorimetric sensorarray, conductive polymer or conductive polymer composite arrays, masssensitive piezoelectric sensors, surface acoustic wave (SAW)transducers, quartz crystal microbalances, functionalized carbonnanotubes and gold nanoparticles based sensors.

Initial work in the field of artificial noses was conducted by Wilkensand Hatman in 1964, though the bulk of research done in this area hasbeen carried out since the early 1980's. See, e.g., W. F. Wilkens and J.D. Hartman, Ann. N.Y. Acad. Sci., 116, 608 (1964); K. Pursaud and G. H.Dodd, Nature, 299, 352-355 (1982); and J. W. Gardner and P. N. Bartlett,Sens. Actuators, B, 18, 210-211 (1994). Vapor-selective detectors or“artificial noses” are typically based upon the production of aninterpretable signal or display upon exposure to a vapor-emittingsubstance or odorant (hereinafter sometimes referred to as an“analyte”). More specifically, typical artificial noses are based uponselective chemical binding or an interface between a detecting elementof the artificial nose and an analyte or odorant, and then transducingof that chemical binding into a signal or display, i.e., signaltransduction.

Conductive polymer composite arrays have been used for artificial noses.That is, a series of chemically-diverse polymers or polymer blends arechosen so that their composite response distinguishes a given odorant oranalyte from others. Examples of polymer array vapor detectors,including conductive polymer and conductive polymer/carbon blackcomposites, are discussed in: M. S. Freund and N. S. Lewis, Proc. Natl.Acad. Sci., U.S.A. 92, 2652-2656 (1995); B. J. Doleman, R. D. Sanner, E.J. Severin, R. H. Grubbs, N. S. Lewis, Anal. Chem., 70, 2560-2564(1998); T. A Dickinson, J. White, J. S. Kauer, D. R. Walt, Nature, 382,697-700 (1996) (polymer array with optical detection); A. E. Hoyt, A. J.Ricco, H. C. Yang, R. M. Crooks, J. Am. Chem. Soc., 117, 8672-8673(1995); and J. W. Grate and M. H. Abraham, Sens. Actuators, B, 3, 85-111(1991).

Other sensing materials include functionalized self-assembled monolayers(SAM), metal oxides, and dendrimers. Signal transduction is commonlyachieved with mass sensitive piezoelectric substrates, surface acousticwave (SAW) transducers, or conductive materials. Optical transducers(based on absorbance or luminescence) have also been examined. Examplesof metal oxide, SAM, and dendrimer-based detectors are discussed in J.W. Gardner, H. V. Shurmer, P. Corcoran, Sens. Actuators, B, 4, 117-121(1991); J. W. Gardner, H. V. Shurmer, T. T. Tan, Sens. Actuators, B, 6,71-75 (1992); and R. M. Crooks and A. J. Ricco, Acc. Chem. Res., 31,219-227 (1998).

Techniques have also been developed using metalloporphyrins for opticaldetection of a specific, single gas such as oxygen or ammonia, and forvapor detection by chemically interactive layers on quartz crystalmicrobalances. See, A. E. Baron et al., Rev. Sci. Instrum., 64,3394-3402 (1993); J. Kavandi et al., Rev. Sci. Instrum., 61, 3340-3347(1990); W. Lee, et al., J. Mater. Chem., 3, 1031-1035 (1993); A. A.Vaughan, M. G. Baron, R. Narayanaswamy, Anal. Comm., 33, 393-396 (1996);J. A. J. Brunink, et al., Anal. Chim. Acta, 325, 53-64 (1996); C.DiNatale, et al., Sens. Actuators, B, 44, 521-526 (1997); and C.DiNatale, et al., Mat. Sci. Eng. C, 5, 209-215 (1998).

Other techniques include functionalized carbon nanotubes integrated intoa transistor. See, DNA-Decorated Carbon Nanotubes for Chemical Sensingby C. Staii and A. T. Johnson, Nano Lett. 5, 1774-1778 (2005); andfunctionalized gold nanoparticles based sensor by G. Peng et al., Nat.Nanotechnol. 4, 669-673 (2009). Also, see nanomaterial-based sensors fordetection of disease by volatile organic compounds by Y. Y. Broza and H.Haick Nanomedicine, 8, 785-806 (2013); and Sniffing the Unique “OdorPrint” of Non-Small-Cell Lung Cancer with Gold Nanoparticles by O.Barash, N. Peled, F. R. Hirsch, H. Haick, Small, 5, 2618-2624 (2009).

Artificial noses based on colorimetric sensor arrays exist that arecapable of detecting volatile organic compounds (VOCs) at lowconcentrations and a high degree of accuracy. Colorimetric sensor arraysthat are capable of detecting VOCs typically contain chemicallyresponsive indicators that change color when VOCs contact the indicatormolecules. The sensing elements of the array, chromogenic indicators,are immobilized in host materials to protect and support the indicatorsonto a solid substrate. Colorimetric sensor arrays are typicallyimmobilized in organically modified silanes (ormosils) or polymers. Fordye-based sensor arrays based on a semi-fluidic polymer, see U.S. Pat.No. 6,368,558, issued on Apr. 9, 2002 and titled “ColorimetricArtificial Nose Having an Array of Dyes and Method for ArtificialOlfaction” by K. S. Suslick and N. A. Rakow. This technology usesmolecular indicators solvated in semi-fluidic plasticizers and porousfilms to make the colorimetric sensor array. There are a severaldrawbacks to using this method for many indicators used in thecolorimetric sensor array. The printed dyes are solvated in asemi-fluidic plasticizer, so the dyes may be unstable and leach intoanalyte solutions, resulting in limited shelf-life and operational life.More recently, nanoporous pigment based colorimetric sensor array havebeen reported to immobilize the indicators in sol-gel matrices. Thissol-gel approach allows customized sol-gel matrices with a range ofhydrophobicity, porosity and surface area. However, this sol-geltechnique does not work with all indicators due to their intrinsicsolubility or pH requirement. For more details, see an OptoelectronicNose for the Detection of Toxic Gases by S. H. Lim et al., Nat. Chem.,1, 562-567 (2009).

SUMMARY

The present invention comprises a colorimetric sensor array comprised ofindicators immobilized in various host materials, which in combinationprovides improved colorimetric sensor arrays for detecting andidentifying various fluidic analytes in both liquid and vapor phase. Inparticular, the invention provides an optimal host material for eachindicator to enhance the array sensitivity and improve the shelf-life ofthe array.

In one aspect, the invention provides a colorimetric sensor arrayinclude a substrate, a first spot on the substrate and a second spot onthe substrate. The first spot includes a first indicator immobilized ina first host material. The second spot includes a second indicator thatmay include a second host material. The composition of the first spot isdifferent from the second spot, which may differ in the indicator and/orthe host material.

In another aspect, the invention provides a method of detecting analytein a sample, including obtaining a first image of the colorimetricsensor array in the absence of the analyte, obtaining a second image ofthe colorimetric sensor array in the presence of the analyte, andanalyzing a color difference between the first image and the secondimage. The resulting pattern of color change manifested by the sensorarray is indicative of a specific or given analyte.

In one embodiment, a sensor array comprises a first and second indicatordeposited on a substrate in a predetermined pattern. The first andsecond indicators can be nucleophilic indicators that have a distinctspectral response to at least one distinct analyte.

The at least one distinct analyte can be in a solution when the firstand second indicators are exposed to the analyte. Both indicators have adistinct spectral response to this analyte. Alternatively, the at leastone distinct analyte can be a volatile organic compound or mixtures ofvolatile organic compounds. The at least one distinct analyte can alsobe any one of, or a combination of, the following: a cancer biomarker,an aldehyde, an organophosphate, an electrophilic analyte, and akeytone.

The first and second indicator demonstrate a distinct spectral responsewhen exposed to any of those possible analytes. The response can be inan ultraviolet range.

The first and second indicator can have a nucleophilic die comprised ofa plasticizer, a sol-gel, or a polymer.

In another embodiment, the present disclosure provides a method ofdetecting an analyte. The method comprises first providing a sensorarray which comprises a first and second indicator deposited on asubstrate in a predetermined pattern. The first and second indicatorsare nucleophilic indicators and have a distinct spectral response todistinct analytes. Then the method comprises exposing the sensor arrayto the analyte and detecting a spectral response after exposing thesensor array to the analyte. The method then comprises correlating thespectral response to the presence of the analyte.

In performing this method, the analyte can be a volatile organiccompound. The analyte can also be in a solution when it is exposed tothe sensor array. Alternatively, when the sensor array is exposed to theanalyte, the sensor array can be exposed to a gas that contains theanalyte. The analyte in the gas can be a volatile organic compound. Inanother embodiment, when the sensor array is exposed to the analyte, thesensor array can be exposed to a liquid that contains the analyte.

The spectral response of the sensor array to the analyte can be in anultraviolet range.

In another embodiment, the present disclosure provides for a sensorarray comprising a first indicator and a second indicator deposited on asubstrate in a predetermined pattern. The first and second indicatorscomprise nucleophilic indicators.

In another embodiment, the present disclosure provides for a method ofdetermining if a patient has a malady. The method comprises firstproviding a sensor array, wherein the sensor array comprises a firstindicator and a second indicator deposited on a substrate in apredetermined pattern. The first and second indicators are nucleophilicindicators. Then the method comprises exposing the sensor array to asample from a patient. Then the method comprises detecting a spectralresponse after exposing the sensor array to the sample. This response iscorrelated to the spectral response that shows in the presence of amalady.

The malady can be any one of, or a combination of, the following:cancer, colon cancer, and sepsis. The sample can be the exhaled breathof a patient. The sample can also be a urine sample. Additionally,exposing the sensor array to a sample from the patient can compriseexposing the sensor array to the headspace gas of a blood sample.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, exemplify the embodiments of the presentinvention and, together with the description, serve to explain andillustrate principles of the invention. The drawings are intended toillustrate major features of the exemplary embodiments in a diagrammaticmanner. The drawings are not intended to depict every feature of actualembodiments nor relative dimensions of the depicted elements, and arenot drawn to scale.

FIG. 1 depicts an example diagram of a system for detecting an arrayresponse;

FIG. 2 depicts a perspective view of an example container and sensorarray;

FIG. 3 depicts a diagram of an example sensor array;

FIG. 4 depicts an example of a method determining a likelihood a patienthas a malady based on detecting and processing a sensor response;

FIG. 5, including FIGS. 5A, 5B, and 5C, depicts illustrations ofdiagrams of an example sensor array after exposure to analytes. FIG. 5Adepicts an example sensor array prior to exposure to an analyte. FIG. 5Bdepicts an example sensor array after to exposure to an analyte. FIG. 5Cdepicts an illustration of a difference map (not based on actualsample).

FIG. 6 depicts an example of a disposable cartridge with a sensor arrayinside;

FIG. 7 depicts another example of a sensor response difference map inRGB and UV (although depicted greyscale);

FIG. 8 depicts two bar graphs showing percentage color change of twonucleophilic indicators for various analytes;

FIG. 9 depicts an example of a microfluidic cartridge with thecolorimetric sensor array printed inside;

FIG. 10 depicts an example of a sensor response difference map fordifferent liquid samples;

FIG. 11 depicts for bar graphs showing percentage color change tovarious electrophilic analytes;

FIG. 12 depicts two example of two sensor response difference maps thatwere captured from sensor arrays using the same indicators on differentsubstrates;

FIG. 13 depicts two graphs showing sensor response to variousconcentrations of formaldehyde.

FIG. 14 depicts two graphs showing the log of the sensor response forthe same data of FIG. 13.

In the drawings, the same reference numbers and any acronyms identifyelements or acts with the same or similar structure or functionality forease of understanding and convenience. To easily identify the discussionof any particular element or act, the most significant digit or digitsin a reference number refer to the Figure number in which that elementis first introduced.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Szycher's Dictionary of MedicalDevices CRC Press, 1995, may provide useful guidance to many of theterms and phrases used herein. One skilled in the art will recognizemany methods and materials similar or equivalent to those describedherein, which could be used in the practice of the present invention.Indeed, the present invention is in no way limited to the methods andmaterials specifically described.

In some embodiments, properties such as dimensions, shapes, relativepositions, and so forth, used to describe and claim certain embodimentsof the invention are to be understood as being modified by the term“about.”

Artificial Nose Technology

VOC selective detectors or “artificial noses” have developed to detectand characterize gaseous samples. A multitude of technologies haveimplemented artificial nose functions including, but not limited to:colorimetric sensor arrays, polymer arrays, mass sensitive piezoelectricsubstrates, surface acoustic wave (SAW) transducers, quartz crystalmicrobalances, functionalized carbon nanotubes and gold nanoparticles.

Initial work in the field of artificial noses was conducted by Wilkensand Hatman in 1964, though the bulk of research done in this area hasbeen carried out since the early 1980's. See, e.g., W. F. Wilkens, A D.Hatman. Ann. NY Acad. Sci., 116, 608 (1964); K. Pursaud, G. H. Dodd.Nature, 299, 352-355 (1982); and J. W. Gardner, P. N., Bartlett. Sensorsand Actuators B, 18-19, 211-220 (1994). Vapor-selective detectors or“artificial noses” are typically based upon the production of aninterpretable signal or display upon exposure to a vapor-emittingsubstance or odorant (hereinafter sometimes referred to as an“analyte”). More specifically, typical artificial noses are based uponselective chemical binding or other molecular interactions in theinterface between detecting a compound of the artificial nose and ananalyte or odorant, and then transforming that chemical binding into asignal or display, i.e., signal transduction.

Polymer arrays having a single indicator have been used for artificialnoses. That is, a series of chemically-diverse polymers or polymerblends are chosen so that their composite response distinguishes a givenodorant or analyte from others. Examples of polymer array vapordetectors, including conductive polymer and conductive polymer/carbonblack composites, are discussed in: M. S. Freund, N. S. Lewis, Proc.Natl. Acad. Sci. USA 92, 2652-2656 (1995); B. J. Doleman, R. D. Sanner,E. J. Severin, R. H. Grubbs, N. S. Lewis, Anal. Chem. 70, 2560-2564(1998); T. A Dickinson, J. White, J. S. Kauer, D. R. Walt, Nature 382,697-700 (1996) (polymer array with optical detection); A E. Hoyt, A J.Ricco, H. C. Yang, R. M. Crooks, J. Am. Chem. Soc. 117, 8672 (1995); andJ. W. Grate, M. H. Abraham, Sensors and Actuators B 3, 85-111 (1991).

Other interface materials include functionalized self-assembledmonolayers (SAM), metal oxides, and dendrimers. Signal transduction iscommonly achieved with mass sensitive piezoelectric substrates, surfaceacoustic wave (SAW) transducers, or conductive materials. Opticaltransducers (based on absorbance or luminescence) have also beenexamined. Examples of metal oxide, SAM, and dendrimer-based detectorsare discussed in J. W. Gardner, H. V. Shurmer, P. Corcoran, Sensors andActuators B 4, 117-121(1991); J. W. Gardner, H. V. Shurmer, T. T. Tan,Sensors and Actuators B 6, 71-75 (1992); and R. M. Crooks, A. J. Ricco,Acc. Chem. Res. 31, 219-227 (1998). These devices also use a singleindicator.

Techniques have also been developed using a metalloporphyrin for opticaldetection of a specific, single gas such as oxygen or ammonia, and forvapor detection by chemically interactive layers on quartz crystalmicrobalances. See A. E. Baron, J. D. S. Danielson, M. Gonterman, J. R.Wan, J. B. Callis, Rev. Sci. Instrum. 64, 3394-3402 (1993); J. Kavandi,et al., Rev. Sci. Instrum. 61, 3340-3347 (1990); W. Lee, et al., J.Mater. Chem. 3, 1031-1035 (1993); A. A. Vaughan, M. G. Baron, R.Narayanaswamy, Anal. Comm. 33, 393-396 (1996); J. A. J. Brunink, et al.,Anal. Chim. Acta 325, 53-64 (1996); C. DiNatale, et al., Sensors andActuators B 44, 521-526 (1997); and C. DiNatale, et al., Mat. Sci. Eng.C 5, 209-215 (1998).

Other techniques include functionalized carbon nanotubes sometimesintegrated into a transistor, see DNA-Decorated Carbon Nanotubes forChemical Sensing Cristian Staii and Alan T. Johnson, Jr., Nano Letters5, 1774-1778 (2005) and functionalized gold nanoparticles see Broza, Y.Y., and Haick, H. Nanomaterial-based sensors for detection of disease byvolatile organic compounds. Nanomedicine, 8(5), 785-806 (2013); Barash,O., Peled, N., Hirsch, F. R., and Haick, H. Sniffing the Unique “OdorPrint” of Non-Small-Cell Lung Cancer with Gold Nanoparticles. Small,5(22), 2618-2624 (2009).

Colorimetric Sensor Arrays

Artificial noses based on colorimetric sensor arrays exist that arecapable of detecting VOCs at low concentrations and a high degree ofaccuracy. Colorimetric sensor arrays may detect VOCs by reacting withthe compounds and changing color based on the amount and type compoundsexposed to the array. The resulting pattern of color changes comprises ahigh-dimensional fingerprint which enables the identification of complexmixtures, including disease signatures in exhaled breath and inheadspace of sealed assays. Various colorimetric sensor arrays aredescribed in the following patent publications to Suslick et al. and allof which are incorporated by reference herein in their entirety: U.S.Pat. No. 6,368,558 to Suslick, U.S. Pat. No. 6,495,102, to Suslick, etal., U.S. Pat. No. 7,261,857, to Suslick et al., and U.S. PatentPublication 2008/0199904.

Chemo-Responsive Indicators

Colorimetric sensor arrays utilizing chemo-responsive (chemicallyresponsive) indicators are capable of detecting individual VOC's andcomplex VOC mixtures down to low part per billion (ppb) concentrations[10, 11, 25]. “Chemo-responsive indicators” refers to any material thatabsorbs, reflects, and/or emits light when exposed to electromagneticradiation, or any other indicator that undergoes a change in spectralproperties in response to certain changes in its chemical environment.“Change in spectral properties” generally refers to a change in thefrequency and/or intensity of the light the colorant absorbs and/oremits. Chemo-responsive indicators may include dyes and pigments.

For example, the following five classes of chemically-responsiveindicators may be utilized: (i) metal-ion-containing indicators thatrespond to Lewis basicity (i.e., electron pair donation, metal ionligation), (ii) pH indicators that respond to Brønsted acidity/basicity(i.e., proton acidity and hydrogen bonding), (iii) indicators with largepermanent dipoles (e.g., solvatochromic dyes) that respond to molecularpolarity, (iv) metal salts that respond to chelation, and (v) redoxindicators that monitor redox properties. Utilizing of this broadspectrum of highly sensitive chemical interactions allows a colorimetricsensor array to detect and identify very diverse classes of metabolitecompounds.

Porphyrins/Metalloporphyrins

For example, for recognition of analytes with Lewis acid/basecapabilities, the use of porphyrins and their metal complexes isdesirable. Metalloporphyrins are ideal for the detection ofmetal-ligating vapors because of their open coordination sites for axialligation, their large spectroscopic shifts upon ligand binding, theirintense coloration, and their ability to provide ligand differentiationbased on metal-selective coordination. Furthermore, metalloporphyrinsare cross-responsive indicators, showing responses to a large variety ofdifferent analytes to different degrees and by different color changes.

Lewis Acid/Base Indicators

A Lewis acid/base indicator is defined as an indicator which has beenidentified for its ability to interact with analytes by acceptor-donorsharing of a pair of electrons from the analyte. This results in achange in color and/or intensity of color that indicates the presence ofthe analyte. Lewis acid/base indicators include metal ion-containing orthree-coordinate boron-containing indicators. The change in spectralproperties for a Lewis acid-base indicator may be related to Lewisacid-base interaction and ligand binding, but also to π-π complexation,hydrogen bonding, and/or polarity changes.

Exemplary Lewis acids include, but are not limited to, metalion-containing porphyrins (i.e., metalloporphyrins), salen complexes,chlorins, bispocket porphyrins, and phthalocyanines. Diversity withinthe metalloporphyrins can be obtained by variation of the parentporphyrin, the porphyrin metal center, or the peripheral porphyrinsubstituents. The parent porphyrin is also referred to as a free baseporphyrin, which has two central nitrogen atoms protonated (i.e.,hydrogen cations bonded to two of the central pyrrole nitrogen atoms).In one example, a parent porphyrin is the so-called free base form5,10,15,20-tetraphenylporphyrin (H₂TPP), its dianion is5,10,15,20-tetraphenyl-porphyrinate(-2) (TPP dianion), its metalatedcomplexes, and its acid forms (H₃TPP⁺ and H₄TPP⁺²). This porphyrin mayform metalated complexes, for example, with Sn⁴⁺, Co³⁺, CO²⁺, Cr³⁺,Mn³⁺, Fe³⁺, Cu²⁺, Ru²⁺, Zn²⁺, Ag²⁺, In³⁺, and Ir³⁺.

Metal ion-containing metalloporphyrin indicators are described, forexample, in U.S. Pat. No. 6,368,558 to Suslick et al. and in U.S. PatentApplication Publication No. 2003/0143112 to Suslick et al., both ofwhich are incorporated by reference herein. Particularly suitable metalions complexed with indicators for detecting ammonia include Zn(II) andCo(III) metals. In particular embodiments of the present invention, theLewis acid indicator is a metalloporphyrin. For example, diversitywithin the metalloporphyrins can be obtained by variation of the parentporphyrin, the porphyrin metal center, or the peripheral porphyrinsubstituents. The parent porphyrin is also referred to as a free baseporphyrin, which has two central nitrogen atoms protonated (i.e.,hydrogen cations bonded to two of the central pyrrole nitrogen atoms). Aparticularly suitable parent porphyrin is5,10,15,20-tetraphenylporphyrinate(-2) (TPP dianion), its metalatedcomplexes, its so-called free base form (H₂TPP) and its acid forms(H₃TPP⁺ and H₄TPP⁺²). Suitable metal ion-containing metalloporphyrinindicators for use in the apparatus and method of the present inventioninclude, but are not limited to:

-   2,3,7,8,12,13,17,18-octafluoro-5,10,15,20-tetrakis-(pentafluorophenyl)porphyrinatocobalt(II)    [Co(F28TPP)];-   2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetraphenylporphyrinatozinc(II)    [Zn(BrsTPP)];-   5,10,15,20-tetraphenylporphyrinatozinc(II) [ZnTPP];-   5(phenyl)-10,15,20-trikis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)    porphyrinatozinc(II) [Zn(Si₆PP)];-   5,10,15,20-tetrakis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrinatozinc(II)    [Zn (Si₈PP];-   5,10,15,20-Tetraphenyl-porphyrinatocobalt (II) [CoTPP];-   5,10,15,20-Tetrakis(2,6-difluorophenyl)porphyrinatozinc(II)    [ZnF₂PP]; and-   5,10,15,20-Tetrakis(2,4,6-trimethylphenyl)porphyrinatozinc(II)    [ZnTMP].

The synthesis of such porphyrins is described in U.S. patent applicationSer. No. 10/279,788.

pH Sensitive Indicators

The chemoresponsive indicator may be, for example, a pH sensitiveindicator. Indicators that are pH sensitive include pH indicator oracid-base indicator dyes that may change color upon exposure to acids orbases. A Brønsted acid indicator of the present disclosure is a pHindicator dye which changes color in response to changes in the proton(Brønsted) acidity or basicity of the environment. For example, Brønstedacid indicators are, in general, non-metalated indicators that areproton donors which can change color by donating a proton to a Brønstedbase (i.e., a proton acceptor). Brønsted acid indicators include, butare not limited to, protonated, but non-metalated, porphyrins, chlorins,bispocket porphyrins, phthalocyanines, and related polypyrrolicindicators. Polypyrrolic indicators, when protonated, are in generalpH-sensitive indicators (i.e., pH indicator or acid-base indicator dyesthat change color upon exposure to acids or bases).

In one embodiment, a Brønsted acid indicator is a non-metalatedporphyrin such as5,10,15,20-tetrakis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrindication [H₄Si₈PP]⁺²; 5,10,15,20-Tetraphenyl-21H,23H-porphine [H₂TPP];or 5,10,15,20-Tetraphenylporphine dication [H₄TPP]⁺². In anotherembodiment of the instant invention, selected Brønsted acid indicatorsinclude, but are not limited to, Bromocresol Purple, Cresol Red, CongoRed, Thymol Blue, Bromocresol Green, Nile Red, Bromothymol Blue, MethylRed, Nitrazine Yellow, Phenol Red, Bromophenol Red, Disperse Orange 25,and Bromophenol Blue. As will be appreciated by the skilled artisan, theBrønsted acids disclosed herein may also be considered Brønsted basesunder particular pH conditions. Likewise, a non-metalated,non-protonated, free base form of a porphyrin may also be considered aBrønsted base. However, these indicator forms are also expresslyconsidered to be within the scope of the indicators disclosed herein.

Examples of Brønsted acid indicators include protonated, butnon-metalated, porphyrins; chlorines; bispocket porphyrins;phthalocyanines; and related polypyrrolic indicators. Examples ofnon-metalated porphyrin Brønsted acid indicators include5,10,15,20-tetrakis(2′,6′-bis(dimethyl-t-butylsiloxyl)phenyl)porphyrindication; 5,10,15,20-tetraphenyl-21H,23H-porphyrin; or5,10,15,20-tetraphenylporphyrin dication. Other examples of Brønstedacid indicators include Chlorophenol Red, Bromocresol Green, BromocresolPurple, Bromothymol Blue, Bromopyrogallol Red, Pyrocatechol Violet,Phenol Red, Thymol Blue, Cresol Red, Alizarin, Mordant Orange, MethylOrange, Methyl Red, Congo Red, Victoria Blue B, Eosin Blue, Fat Brown B,Benzopurpurin 4B, Phloxine B, Orange G, Metanil Yellow, Naphthol GreenB, Methylene Blue, Safranine O, Methylene Violet 3RAX, Sudan Orange G,Morin Hydrate, Neutral Red, Disperse Orange #25, Rosolic Acid, Fat BrownRR, Cyanidin chloride, 3,6-Acridineamine,6′-Butoxy-2,6-diamino-3,3′-azodipyridine, para-Rosaniline Base, AcridineOrange Base, Crystal Violet, Malachite Green Carbinol Base, Nile Red,Nile Blue, Nitrazine Yellow, Bromophenol Red, Bromophenol Blue,Bromoxylenol Blue, Xylenol Orange Tetrasodium Salt,1-[4-[[4-(dimethylamino)phenyl]azo]phenyl]-2,2,2-trifluoro-ethanone-,4-[2-[4-(dimethylamino)phenyl]ethenyl]-2,6-dimethyl-pyryliumperchlorate, and 1-amino-4-(4-decylphenylazo)-naphthalene.

Solvachromatic Dyes

The chemoresponsive dye may be, for example, a solvatochromic dye or avapochromic dye. Solvatochromic dyes that may be utilized change colorin response to changes in the general polarity of their environment,primarily through strong dipole-dipole and dispersion interactions. Tosome extent, all dyes inherently are solvatochromic, with some beingmore responsive than others. Particular examples of suitablesolvatochromic dyes include, but are not limited to Reichardt's dyes,Nile Red, Disperse Orange #25, Disperse Orange #3, Phenol Blue,Merocyanine 540, 1-Ethyl-4-(2-hydroxystyryl)pyridinium iodide,4-hydroxystyryl-pyridinium dye, 4-methoxycarbonyl-1-ethylpyridiniumiodide, and 2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)-phenolate.

The addition of at least one Brønsted acid dye to an array containing atleast one metal ion-containing Lewis acid dye can improve thesensitivity of the array for particular analytes and increase theability to discriminate between analytes. For example, colorimetricsensor arrays have been shown to detect volatile organic compounds andcomplex mixtures down to ppb levels (Rakow, et al. (2005) Angew. Chem.Int. Ed. 44:4528-4532). Further, the use of one or more metalion-containing dyes in combination with one or more Brønsted acid dyescan advantageously create a signature indicative of the presence of aparticular analyte. Thus, while some embodiments may utilize at leastone Lewis acid and/or base dye, one Brønsted acidic and/or basic dye, orone zwitterionic solvatochromic dye, other embodiments of thisdisclosure may utilize use at least two different classes of dyes on theinstant arrays. In one embodiment, the colorimetric sensor arraycontains at least one Lewis acid and/or base dye, one Brønsted acidicand/or basic dye, or one zwitterionic solvatochromic dye. In anotherembodiment, the colorimetric sensor array contains at least one Lewisacid and/or base dye and one Brønsted acidic and/or basic dye. In afurther embodiment, the colorimetric sensor array contains at least oneLewis acid and/or base dye and one zwitterionic solvatochromic dye. Inyet a further embodiment, the colorimetric sensor array contains atleast one Brønsted acidic and/or basic dye and one zwitterionicsolvatochromic dye. Still further embodiments may utilize at least threedifferent classes of dyes on the instant arrays, i.e., at least oneLewis acid and/or base dye, one Brønsted acidic and/or basic dye, andone zwitterionic solvatochromic dye.

An array that includes a pH sensitive dye and/or a solvatochromic orvapochromic dye may be useful in differentiating analytes that do notbind to, or bind only weakly to, metal ions. Such analytes includeacidic compounds, such as carboxylic acids, and certain organiccompounds lacking ligatable functionality. Examples of organic compoundslacking ligatable functionality include simple alkanes, arenes, and somealkenes and alkynes, especially if sterically hindered. Examples oforganic compounds lacking ligatable functionality also include moleculesthat are sufficiently sterically hindered to preclude effectiveligation. Arrays that include a pH sensitive and/or a solvatochromic orvapochromic dye are described, for example, in U.S. Patent ApplicationPublication No. 2003/0143112 to Suslick et al., which is incorporated byreference herein.

Redox Sensitive Indicators

The chemoresponsive indicator may be, for example, a redox sensitiveindicator that undergoes a change in spectral properties depending uponits oxidation state. Examples of dyes that are redox sensitive includeredox indicators such as methylene blue, naphthol blue-black, brilliantponceau, alpha-naphthoflavone, basic fuchsin, quinoline yellow, thioninacetate, methyl orange, neutral red, diphenylamine,diphenylaminesulfonic acid, 1,10-phenanthroline iron(II), permanganatesalts, silver salts, and mercuric salts.

Metal Ion Sensitive Indicators

The chemoresponsive indicator may be, for example, a metal ion sensitiveindicator that undergoes a change in spectral properties in the presenceof metal ions. Examples of dyes that are metal ion sensitive includemetal ion indicators, such as eriochrome black T, murexide,1-(2-pyridylazo)-2naphthol, and pyrocatechol violet.

Deficiencies of Existing Arrays

As disclosed herein, existing arrays do not contain sufficientindicators for detecting electrophilic analytes. For instance, while asingle nucleophilic indicator has been used in an array, that alone hasnot produced an array sensitive enough to detect electrophilic analytes.Particularly, prior arrays did not detect certain aldehydes, esters andketones with a high degree of accuracy or sensitivity.

This is particularly relevant because aldehydes and ketones are usefulanalytes to detect a variety of cancers. Additionally, formaldehydedetection is useful because formaldehyde is a frequent pollutant. Also,organophosphates (esters) are electrophilic compounds used in pesticidesand chemical warfare agents. Accordingly, the use of compounds that candetect electrophilic compounds with a high degree of sensitivity andselectivity would be highly useful.

However, previous efforts have failed to produce an array that coulddetect electrophilic compounds with a high degree of sensitivity andaccuracy. For instance, in some cases, nucleophilic amines (anucleophilic agent) have been used with a pH indicator. Therefore,reactions of nucleophilic agents with electrophilic analytes wouldresult in the change in acidity that can be detected with a pHindicator. However, in those cases the pH indicator would be highlycross-reactive toward acidic analytes, and could not readilydiscriminate between acidic and electrophilic analytes. Thesenucleophilic agents were not sensitive enough primarily because thenucleophilic agent did not contain a chromophore. The chemistry involvedonly a rough indication of pH changes with a secondary pH indicatormolecule.

In a few prior examples, a single nucleophilic indicator (nucleophilicmolecule that includes a chromophore as defined herein) has been used inan array. However, in those cases, the sensitivity and selectivity wasnot satisfactory to be practically useful because the array onlycontained a single nucleophilic indicator.

Arrays with Nucleophilic Indicators

Accordingly, it was investigated as to whether an array of at least twoor more nucleophilic indicators could be sufficiently sensitive to bothdetect and discriminate different kinds of electrophilic analytes betterthan existing nucleophilic agents. Accordingly, it was discovered thatan array of nucleophilic indicators had a surprising sensitivity andselectivity to identify and classify nucleophilic compounds at very lowconcentrations. For instance, it was discovered that formaldehyde couldbe detect at 10 ppb using an array of nucleophilic indicators.

Additionally, it was determined that a nucleophilic array could detectvolatile electrophilic biomarkers for colon cancer in exhaled breath,and organophosphates in liquid form. Accordingly, it was discovered thatusing a multitude of nucleophilic indicators has the sensitivity to moreaccurately detect electrophilic molecules.

The nucleophilic indicators used for these investigations were definedherein as “nucleophilic indicators.” These are defined herein asnucleophilic molecules that include a chromophore. Accordingly, theindicator itself changes color. This is opposed to a nucleophilicagent—a molecule without a chromophore—that undergoes a chemical changethat is detected by a secondary indicator molecule. For instance, somenucleophilic agents exhibit a change in pH when interacting withelectronic rich molecules, and therefore the nucleophilic agent incombination with a pH indicator can detect electrophilic analytes.However, it has been discovered that an array of nucleophilicindicators, which has a chromophore attached to the nucleophilicmolecule itself, is much more sensitive and selective than anucleophilic agent.

Chemoresponsive Pigments

The chemoresponsive indicator may be a chemoresponsive pigment. In somecases, the chemoresponsive pigment is a porous pigment in whichchemoresponsive dyes are immobilized in a porous matrix. A porouspigment particle has a chemoresponsive surface area that is much greaterthan the chemoresponsive surface area of a corresponding nonporouspigment particle. Examples of porous matrices include porous calciumcarbonate, porous magnesium carbonate, porous silica, porous alumina,porous titania, and zeolites.

Chemoresponsive Nanoparticle

The chemoresponsive indicator may be a chemoresponsive nanoparticle. Achemoresponsive nanoparticle may be a discrete nanoparticle, or it maybe formed from nanoparticle-forming ions or molecules. The nanoparticlemay be in a variety of forms, including a nanosphere, a nanorod, ananofiber, and a nanotube. Examples of chemoresponsive nanoparticlesinclude nanoporous porphyrin solids, semiconductor nanoparticles such asquantum dots, and metal nanoparticles.

Array of Indicators

The use of more than one type of chemoresponsive colorant may expand therange of analytes to which the array is sensitive, may improvesensitivity to some analytes, and/or may increase the ability todiscriminate between analytes. In some cases, a colorimetric arrayincludes 2 to 1,000 sensors, 4 to 500 sensors, or 8 to 250 sensors. Incertain cases, a colorimetric array includes from 10 to 100 sensors(e.g., 16 to 80 sensors, 36 sensors, or 60 sensors). Each sensor in acolorimetric array may include a different colorant. However, it may bedesirable to include duplicate sensors that include the same colorant.Duplicate sensors may be useful, for example, to provide redundancy tothe array and/or to serve as an indicator for quality control. Table 1lists exemplary chemoresponsive indicators for a colorimetric sensorarray having 36 sensors.

TABLE 1 Exemplary chemoresponsive colorants for a colorimetric sensorarray. No. Colorant 1 5,10,15,20-Tetraphenyl-21H,23H-porphine zinc (II)2 5,10,15,20-Tetraphenyl-21H,23H-porphine copper (II) 35,10,15,20-Tetraphenyl-21H,23H-porphine manganese (III) chloride 42,3,7,8,12,13,17,18-Octaethyl-21H,23H-porphine iron (III) chloride 55,10,15,20-Tetraphenyl-21H,23H-porphine cobalt (II) 6meso-Tetra(2,4,6-trimethylphenyl)porphine 7 Nitrazine Yellow (basic) 8Methyl Red (basic) 9 Chlorophenol Red (basic) 10 Napthyl Blue Black 11Bromothymol Blue (basic) 12 Thymol Blue (basic) 13 m-Cresol Purple(basic) 14 Zinc (II) acetate with m-Cresol purple (basic) 15 Mercury(II) chloride with Bromophenol Blue (basic) 16 Mercury (II) chloridewith Bromocresol Green (basic) 17 Lead (II) acetate 18Tetraiodophenolsulfonephthalein 19 Fluorescein 20 Bromocresol Green 21Methyl Red 22 Bromocresol Purple 23 Bromophenol Red 24 Brilliant Yellow25 Silver nitrate + Bromophenol Blue (basic) 26 Silver nitrate +Bromocresol Green (basic) 27 Cresol Red (acidic) 28 Disperse Orange 2529 m-Cresol Purple 30 Nitrazine Yellow 31 Cresol Red 32 BromocresolGreen 33 Phenol Red 34 Thymol Blue 35 Bromophenol Blue 36 m-CresolPurple

Table 2 lists example of an array that incorporate nucleophilicindicators for a vapor sensor.

TABLE 2 Exemplary chemoresponsive colorants for a colorimetric sensorarray incorporating nucleophilic indicators for a vapor sensor: No.Colorant 1 Purpald + TBAOH 2 Purpald + TBAOH 3 Fiducial (carbon black) 4Parosoaniline 5 Parosoaniline 6 Acetylacetone +2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride 7 Acetylacetone +2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride 8 Acetylacetone +2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride 9 Acetylacetone +5-aminofluorescein 10 Acetylacetone + 5-aminofluorescein 11Acetylacetone + 5-Aminofluorescein 12 2 4-Dinitrophenylhydrazine + TsOH13 2 4-Dinitrophenylhydrazine 14 2 4-Dinitrophenylhydrazine 15 24-Dinitrophenylhydrazine + 4-Nitroaniline 16 24-Dinitrophenylhydrazine + 4-Nitroaniline 17 24-Dinitrophenylhydrazine + 4-Nitroaniline 183-Methyl-2-benzothiazolinone hydrazine + Fe(NO₃)₃ 193-Methyl-2-benzothiazolinone hydrazine + Fe(NO₃)₃ 203-Methyl-2-benzothiazolinone hydrazine + Pb(OAc)₄ 213-Methyl-2-benzothiazolinone hydrazine + Pb(OAc)₄ 22 Chromotropic acid +Ag₂O 23 Chromotropic acid + Ag₂O 24 Chromotropic acid + Ag₂O 25N-(Rhodamine B)-deoxylactam-ethylenediamine + TBAOH 26 N-(RhodamineB)-deoxylactam-ethylenediamine + TBAOH 27 N-(RhodamineB)-deoxylactam-ethylenediamine + TBAOH 28[4-[(3,5-Dimethyl-1H-pyrrol-2-yl-N)(3,5-dimethyl-2H-pyrrol-2-ylidene-N)methyl]-1,2-benzenediaminato]difluoroboron 29[4-[(3,5-Dimethyl-1H-pyrrol-2-yl-N)(3,5-dimethyl-2H-pyrrol-2-ylidene-N)methyl]-1,2-benzenediaminato]difluoroboron + Sc(OTf)₃ 305-Aminofluorescein 31 4,4-Dimethylaminostyryl pyridine 324,4-Dimethylaminostyryl pyridine

Table 3 lists example of an array that incorporate nucleophilicindicators for a liquid sensor.

TABLE 3 Exemplary chemoresponsive colorants for a colorimetric sensorarray incorporating nucleophilic indicators for a liquid sensor: No.Colorant 1 Ethyl bis(2,4-dinitrophenyl) acetate 2 Yellow dye A2 3 Reddye E 4 Nitrazine Yellow 5 Methyl Red + TBAOH 6Tetraiodophenolsulfonephthalein 7 Bromocresol Green 8[4-[3,5-dimethyl-1H-pyrrol-2-yl-N)(3,5-dimethyl-2H-pyrrol-2-ylidene-N)methyl]-1,2-benzenediaminato]difluoroboron + NaOH 9 Fiducial(carbon black) 10 (E)-5,5-Difluoro-7-(4-([2-(2-hydroxyethoxy)ethyl]-(methyl)amino)styrl)-1,3,9-trimethyl-10-phenyl-5H-dipyrrolo(1,2-c:2′,1′-f](1,3,2)diazaborinin-4-ium-5-uide - Europium(III)complex + NaOH 11 (E)-5,5-Difluoro-7-(4-([2-(2-hydroxyethoxy)ethyl]-(methyl)amino)styrl)-1,3,9-trimethyl-10-phenyl-5H-dipyrrolo(1,2-c:2′,1′-f](1,3,2)diazaborinin-4-ium-5-uide + NaOH 12Co(II)TPP + Bromocresol purple 13 Zn(II)TPP + Bromophenol blue 14Pyrocatechol violet + TBAOH 15 2,6-dichloroindophenol sodium salthydrate + TBAOH 16 Sn(IV)TPPCl₂ 17 Fiducial (carbon black) 18 HgCl₂ +Bromocresol Green + TBAOH 19 LiNO₃ + Cresol Red + TBAOH 205-Aminofluorescein 21 Co(II)TPP 22 N-(rhodamineB)-deoxylactam-ethylenediamine + TBAOH 23 Mg(II)TPP 241-Methyl-2-phenylidone 25 Fiducial (carbon black) 26 Mn(III)TPPCl 27Fe(III)OEPCl 28 Rh(III)TPPCl 29 Zn(II)TMP 30 4-Aminophenylsulfone 31Copper (II) neodecanoate 32 4-(4-Nitrobenzyl)pyridine + N-Benzylaniline

Table 4 lists example of an array that incorporate nucleophilicindicator indicators for a vapor sensor.

TABLE 4 Exemplary chemoresponsive colorants for a colorimetric sensorarray incorporating nucleophilic indicators for a vapor sensor: No.Colorant 1 Ethyl bis(2,4-dinitrophenyl) acetate 2 Yellow dye A2 3 Reddye E 4 Nitrazine Yellow 5 Methyl Red + TBAOH 6Tetraiodophenolsulfonephthalein 7 Bromocresol Green 8[4-[3,5-dimethyl-1H-pyrrol-2-yl-N)(3,5-dimethyl-2H-pyrrol-2-ylidene-N)methyl]-1,2-benzenediaminato]difluoroboron + NaOH 9 Fiducial(carbon black) 10 (E)-5,5-Difluoro-7-(4-([2-(2-hydroxyethoxy)ethyl]-(methyl)amino)styrl)-1,3,9-trimethyl-10-phenyl-5H-dipyrrolo(1,2-c:2′,1′-f](1,3,2)diazaborinin-4-ium-5-uide - Europium(III)complex + NaOH 11 (E)-5,5-Difluoro-7-(4-([2-(2-hydroxyethoxy)ethyl]-(methyl)amino)styrl)-1,3,9-trimethyl-10-phenyl-5H-dipyrrolo(1,2-c:2′,1′-f](1,3,2)diazaborinin-4-ium-5-uide + NaOH 12Co(II)TPP + Bromocresol purple 13 Zn(II)TPP + Bromophenol blue 14Pyrocatechol Violet + TBAOH 15 2,6-Dichloroindophenol sodium salthydrate + TBAOH 16 Sn(IV)TPPCl₂ 17 Fiducial (carbon black) 18 HgCl₂ +Bromocresol Green + TBAOH 19 LiNO₃ + Cresol Red + TBAOH 205-Aminofluorescein 21 Co(II)TPP 22 N-(rhodamineB)-deoxylactam-ethylenediamine + TBAOH 23 Mg(II)TPP 241-Methyl-2-phenylidone 25 Fiducial (carbon black) 26 Mn(III)TPPCl 27Fe(III)OEPCl 28 Rh(III)TPPCl 29 Zn(II)TMP 30 4-Aminophenylsulfone 31Copper (II) neodecanoate 32 4-(4-Nitrobenzyl)pyridine + N-Benzylaniline

Indicator Formulation

Indicators may be made with any number of formulations. For instances,some indicators may be made in plasticizer formulations. Theseformulations are generally liquids and may spread out quite a bit overtime. In other examples, formulations include polymer based formationsthat form a polymeric network to deposit the indicator. For instance,polymer based formulations may include, cellulous, polystyrene,polyvinyl alcohol, poly(methyl methacrylate) or polyvinyl chloride. Inother examples, nanoporous material may be used to form nanoporouspigments. Certain host materials may be more suitable for certainanalytes and/or indicators, depending on affinity, surface area, andinteractions with the indicator or analyte.

The nanoporous material may be used to convert a dye to a nanoporouspigment. The nanoporous pigment may be made from a nanoporous materialand a first immobilized, chemoresponsive colorant. In some examples, anarray will be formed by depositing a first liquid that is a nanoporousmaterial precursor and a chemoresponsive colorant that may be aprepolymer, ceramic precursor or mixtures of these. Then, the nanoporousprecursor may be solidified to form a nanoporous material. The firstnanoporous material precursor may be, for example, a polymer, aprepolymer, ceramic precursors, or mixtures of these. The first liquidmay include other ingredients, such as a solvent and/or a surfactant.

The first nanoporous material precursor may include starting materialsfor a ceramic that have been at least partially hydrolyzed. The firstliquid may be formed by combining ingredients including startingmaterials for a ceramic, a solvent, and the first chemoresponsivecolorant to form a first mixture, and then hydrolyzing the first mixtureto form a sol. Solidifying the first nanoporous material precursor mayinclude condensing the first nanoporous material precursor to form agel, and drying the gel to form the first nanoporous material.

The solidifying may include any method that converts the nanoporousmaterial precursor into a nanoporous material. Examples ofsolidification methods include chemical crosslinking, exposure to UVradiation, and heating. In one example, the solidifying includes heatingthe liquid on the substrate. Initial curing at room temperature for 24to 72 hours may be preferred in order to maintain porosity of thenanoporous pigment. Additional heating may be performed, for example, ina standard convection oven. If the substrate is temperature-sensitive,heating the liquid for 24 hours at temperatures lower than 70° C. ispreferred. When preparing a spot that includes a nanoporous ceramic anda pH responsive indicator as the chemoresponsive colorant, solidifyingat 60° C. or even at room temperature is preferred. With more thermallyrobust substrates, solidifying may be completed much more rapidly, forexample in 1 hour at 120° C.

The nanoporous material may be any material that includes pores,reticulations or void spaces with dimensions from 0.2 to 1000 nm.Preferably the nanoporous material includes pores with dimensions from0.5 to 100 nm. Preferably the nanoporous material includes pores thatare interconnected, such that a fluid can flow between the pores of thematerial. A nanoporous material may be, for example, an inorganicnetwork, such as a porous ceramic or a zeolite. A nanoporous materialmay be, for example, an organic network, such as a collection of carbontubes or a crosslinked gel. A nanoporous material may be, for example, amembrane material, such as a microfiltration membrane or anultrafiltration membrane. A nanoporous material may be a combination ofan inorganic network, an organic network and/or a membrane, such as aninorganic/organic composite.

A nanoporous pigment may be fabricated by the immobilization ofchemically responsive dyes in organically modified siloxanes (ormosils).In some embodiments, the pigment is created by utilizing an electronicspray to generate an aerosol from precursor solutions containing the dyeand other materials, which is then heated to form dye encapsulatedmicrospheres. These dye-encapsulated microspheres can then be printed onpaper, such as chromatography paper to form a colorimetric sensor array.These porous sol-gel ormosils may provide a good matrix for colorantsdue to high surface area, good stability over a wide range of pH,relative inertness in many environments, and transparency in theUV-visible spectrum.

A nanoporous sol-gel matrix has enormous surface area at a microscopicscale, which results in the part-per-billion (ppb) sensitivity. In someembodiments, a nanoporous sol-gel matrix may be required to detect thetrace volatile organic compound (VOC) signatures of lung cancer andother diseases in urine or other biological fluids. The nanoporouspigment is a silicon-based sol-gel with enormous surface area, vastlyincreasing interaction opportunities between analyte and indicator andthereby achieving great sensitivity across a wide range of volatilemolecules, including species crucial for cancer diagnosis. Furthermore,the high chemical resistance of the nanoporous pigment allows amanufacturer to increase the chemical diversity of the dyes deposited onthe nanoporous substrate by adding chromogenic reagents that were tooreactive to incorporate onto substrates comprised of differentmaterials. Nanoporous pigments are more fully described in Lim et al.,Chemically Responsive Nanoporous Pigments: Colorimetric Sensor Arras andthe Identification of Aliphatic Amines, Langmuir 24 (22), 2008, which isincorporated by reference herein in its entirety.

Indicator Formulations—Nucleophilic Indicators

The indicator performance can be substantially influenced by theselection of host materials. For sensing application, the indicator mustbe immobilized in a host material, but accessible to the analyte. Thenonreactive host material must be able to retain the indicator in placeto prevent dye leaching or spreading over time, but porous enough toallow free diffusion of the analyte to the indicator. Furthermore, theindicator needs to be solvated to interact with analytes. When anindicator crystallizes in the host material, the reactivity of theindicator may diminish and only the surface of the crystal will react.Hence, custom formulations suited for different nucleophilic indicatorsare developed, which include polymer based formulations, sol-gel (e.g.nanoporous) formulations, and plasticizer based formulations.Accordingly, each formulation may be customized based on the indicatoror the substrate.

Indicator Substrate

In accordance with the present invention, the plurality ofchemo-responsive indicators may be deposited on an array substrate in apredetermined pattern combination. Alternatively stated, the indicatorsare arranged in a two-dimensional (or linear or other arrangement)spatially-resolved configuration so that upon interaction with one ormore analytes, a distinct color and intensity response of each indicatorcreates a signature indicative of the one or more analytes. A pluralityof chemoresponsive indicators encompasses 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, or 50 individual indicators. In particularembodiments, a plurality of chemo-responsive indicators is 2 or more, 5or more, 10 or more, 15 or more, 20 or more, 25 or more, or 30 or moreindicators. The chemo-responsive indicators can be deposited inpredetermined pattern combinations of rows, columns, spirals, etc., andone or more chemoresponsive indicator arrays can be used in a container.Indicators can be covalently or non-covalently affixed in or on acolorimetric sensor array substrate by direct deposition, including, butnot limited to, pin-printing, airbrushing, ink-jet printing, screenprinting, stamping, micropipette spotting, or nanoliter dispensing.

The substrate for retaining the chemo-responsive indicators may be anysuitable material or materials, including but not limited to,chromatography plates, paper, filter papers, porous membranes, orproperly machined polymers, glasses, or metals. In some embodiments, thesubstrate may be a hydrophobic or hydrophilic substrate.

Exposure of the Array to Analytes

For gas or vapor analytes, a gas stream containing the analyte is passedover the array, and images may be obtained before, during and/or afterexposure to the gas stream. Preferably, an image is obtained after thesample and the array have equilibrated. If the gas stream is notpressurized, it may be useful to use a miniaturized pump or fan.

For analytes dissolved in a solvent, either aqueous or non-aqueous, thefirst image may be obtained in air or, preferably, after exposure to thepure carrier solvent that is used of the sample. The second image of thearray may be obtained after the start of the exposure of the array tothe sample. Preferably an image is obtained after the sample and thearray have equilibrated.

A colorimetric sensor array may be used to detect analytes in exhaledbreath. Detection of compounds in exhaled breath can be useful indetecting infection or disease. The colorimetric detection of ammonia inexhaled breath is described, for example, in U.S. Patent ApplicationPublication No. 2005/0171449 to Suslick et al., which is incorporated byreference herein.

The colorimetric sensor array may be in gaseous or liquid communicationwith a fluidic sample and/or a solid or liquid culture medium, or othermaterials containing the sample. This will allow volatile organiccompounds (VOCs) emitted from the sample (e.g. from microorganisms inthe sample) to evaporate into the headspace of the container and comeinto contact with the colorimetric sensor array. In other examples,analytes will be contained in the liquid and will react with thecolorimetric sensor once they contact the sensor array. In someembodiments, the container is sealed, and the colorimetric sensor arrayis exposed to VOCs emitted from the microorganisms or other sources ofVOCs. In other embodiments, different containers or other mechanismscould be utilized to expose the colorimetric sensor array to gas emittedfrom the sample. This could include various channels or tubing thatcould transport the volatile organic compounds emitted from the sampleinto a gaseous state.

Analyte Detection—Nucleophilic Indicators

Nucleophilic indicators are particularly suited to detecting electrondeficient molecules or electrophilic analytes. For instance, certaintypes of aldehydes, ketones, and esters are particularly well detectedby nucleophilic indicators.

Aldehydes are important for a variety of reasons, including for thedetection of formaldehyde. Formaldehyde occurs in the environment up to0.03 ppm parts of air. Materials that incorporate formaldehyde, such asformaldehyde foam insulation (UFFI), can release it in the form of gasor vapor. For instance, pressed-wood products are a major source offormaldehyde indoor pollution. Cigarette smoke, gas stoves, wood-burningstoves, and kerosene heaters can also release formaldehyde. It is toxicto both plants and animals, and is particularly dangerous for the humaneyes.

Formaldehyde pollution can also be found in water, and originates fromoxidation of organic matter during ozonation and chlorination. Indrinking water, it can arise from leaching from plastic fittings andwater treatment processes.

Accordingly, detection of formaldehyde in gaseous and liquid states isquite important for detection of pollution. However, obtaining apractical method that is sensitive enough can be difficult. See, e.g.Dai, et al. “A simple spot test quantification method to determineformaldehyde in aqueous samples.” Accordingly, the disclosed arrays withnucleophilic indicators provide a much more sensitive and selectivemethod to detect formaldehyde.

Aldehydes (and ketones) are also important class of biomarkers in breathanalysis, including cancer diagnosis. A plethora of aldehydes andketones have been linked to various cancers. However, these biomarkersmay be trace amounts in bodily fluids or exhaled gasses and mixed with aplethora of different signals. Accordingly, developing a test sensitiveenough to detect cancer in a patient requires extraordinary sensitivityand selectivity. As disclosed below, a breath test to detect biomarkersfor cancer using a nucleophilic sensor array has been developed. Table 5illustrates examples of cancer biomarkers that may be detected usingnucleophilic indicators.

TABLE 5 Exemplary disease biomarkers that react with nucleophilicindicators: Chemical Analyte Class Disease Biomarker 1-phenyl- ketonesBreast cancer ethanone 2,3-dihydro- ketones Breast cancer 1-phenyl-4(1H)- quinazolinone 2-butanone ketones Lung cancer, chronic liverdisease 2-pentanone ketones Lung cancer, chronic liver disease3-hydroxy-2- ketones Lung cancer butanone acetone ketones Lung cancer,diabetes/hyperglycemia, chronic liver disease, stomach cancer,gastro-esophageal cancer, homeostatic balance control 2- aldehydesEmphysema methylbutanal acetaldehyde aldehydes Alcoholism,gastro-esophageal cancer, liver disease butanal aldehydes Lung cancerformaldehyde aldehydes Lung cancer, breast cancer, gastro- esophagealcancer furfural aldehydes Gastric ulcer heptanal aldehydes Lung cancer,breast cancer hexanal aldehydes Lung cancer nonanal aldehydes Lungcancer pentanal aldehydes Lung cancer propanal aldehydes Lung cancer

Additionally, nucleophilic indicators may be useful for detection oforganophosphates. These analytes are used in pesticides and insecticide;therefore, the arrays may be useful for detection of pollution. In otherexamples, organophosphates may be utilized for nerve gas or otherchemical weapons. Accordingly, testing for organophosphates may havesome particularly useful military applications.

Detection of Artificial Nose Response Detector

In embodiments where a colorimetric sensor array is utilized as theartificial nose technology, the color changes of the chemicallyresponsive indicators may be detected by any suitable optical or otherdetector. In embodiments pertaining to a colorimetric sensor array, adetector may monitor the spectroscopic response, transmission responseor reflectance response of the indicators on the colorimetric sensingelement at one or more wavelengths in a spatially resolved fashion sothat all of the spots in the colorimetric sensor array are individuallyimaged or addressed and the color of each spot is individuallydetermined. For the purposes of the present disclosure, the terms colorand colorimetric are intended to include wavelengths in the visibleportion of the electromagnetic spectrum, as well as the invisibleportion of the electromagnetic spectrum, e.g., infrared and ultraviolet.

Color detection can be accomplished with an imaging spectrophotometer, aflatbed scanner, slide scanner, a video or CCD or CMOS digital camera,or a light source combined with a CCD or CMOS detector. Any still orvideo as well as analog or digital camera can be employed. Moreover, anyimaging format can be used, e.g., RGB (red, green and blue) or YUV. Evensimple gray scale imaging can be used. In other embodiments utilizingother artificial nose technologies, a detector or sensor may similarlybe used to provide a response of the detector indicative of themolecular interactions occurring at the detector probe or other sensor.

The sensitivity of a colorimetric sensor array is primarily a functionof two factors, the ability of an indicator spot to change color whenexposed to an analyte and the ability of the detector to measure thatcolor change.

In some embodiments, an optical spectroscopic measurement system candivide the visible spectrum into as many as individual bandpass windowswhereas a three-color imaging system by definition contains only threesuch windows. An optical spectroscopic measurement system is thereforecapable of detecting smaller color changes than can be detected bythree-color imaging systems, effectively increasing the sensitivity ofthe entire cross-responsive sensing system. Accordingly, in particularembodiments of the present disclosure, an optical spectroscopicmeasurement system is employed as a detector. As used herein, opticalspectroscopic measurement systems refer to any system that yields highercolor resolution than a three-color imaging system. This can be animaging spectrograph, fiber optic probe(s) coupled to a spectrograph, orother spectroscopic system.

Detectors for Arrays with Nucleophilic Indicators

In some examples, it was determined that a detector capable of sensingultraviolet (“UV”) rays recorded optimal difference in detecting certaintypes of analytes using nucleophilic indicators. For instance,differences in colorimetric response could not be reliably detected inthe RGB range, and instead were only recorded in the UV range.Accordingly, in some examples, a nucleophilic detector and analysis willbe performed in the UV range to find optimal differences.

Detection Process and System Setup

FIG. 1 illustrates an example of a system, in some embodiments, wherethe sample and container 200, as shown in FIG. 2, are maintained at aconstant temperature by an incubator. Referring back to FIG. 1, in someembodiments, the detector 130 may be incorporated into an incubator toallow the detector 130 to continuously, or intermittently record thecolorimetric response of the indicators 710 through a window 270 whileleaving the sample undisturbed at constant temperature. Afterapplication of the VOCs contained in the urine may begin to react withthe colorimetric sensor array 120.

Prior to application of a sample, a detector 130 may record an image ofthe presently loaded sensor array 120 as a control for later comparisonand subtraction of color changes. Accordingly, this will allow thesystem to measure color changes based on variation from that particulararray's initial color profile. In embodiments associated with otherartificial nose technologies, the detector may record an initial readingfor comparison to a later reading after introduction of a sample. Afterapplication of a sample to a sensor container 200, a detector 130 may atvarious intervals or after a set time interval, detect and record thecolorimetric response of the indicators 710 or other detector 130response. In some embodiments, software may be configured on server 150or processing device 140 for automatically controlling the precisetiming of detector 130 and recording of the data captured by detector130. For example, the detector 130 may record an image every minute,every 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes, or at intervals in-between,or at 20 or 30 minutes, or other suitable intervals. In someembodiments, the detector 130 may continuously record data from thecolorimetric sensor array 120. The detector 130 may record images for anhour, 2, 3, 4, 5, 6, hours, or other suitable time frame. In someembodiments, the time frame may be selected based on when the colorchange rate is near homeostasis or has stopped reacting. In otherembodiments, the color change may be stopped when a color change ratedrops below a certain threshold.

FIG. 2 depicts exemplary container 200 with colorimetric sensor array120 for detecting volatile organic compounds emitted from a sample.Container 200 may include a solid or liquid culture medium generallyknown in the art. A sample, such as a fluid sample (e.g., blood, sputum,exhaled breath) from a mammal, a tissue sample from a mammal, or thelike, is placed or injected in container 200.

FIG. 3 illustrates an embodiment of a colorimetric sensor array 120. Inan embodiment, a colorimetric sensor array 120 may include a substrate720 upon which a variety of chemically responsive indicators 710 may bedeposited. The indicators 710 may change color after exposure to andreacting with volatile organic compounds. Certain indicators areresponsive to certain VOCs allowing for a particular mixture of VOCs tobe determined by its unique color change exhibited on the sensor array120.

Data Processing

Data output from the detector 130 or other instruments associated withan artificial nose technology may then be stored and later processed forevaluation and diagnosis of the sample. A detector 130 may beincorporated into any suitable sensor or other instruments associatedwith an artificial nose technology system. The processing of the datamay be performed on the processing device 140, server 150, or othercomputing device connected to the system. Various artificial nosetechnologies and systems may provide a response or an output indicativeof the chemical or molecular interactions occurring at a sensorassociated with the artificial nose technology. For example, manyembodiments may utilize a detector 130 to detect changes afterintroduction of a urine sample containing VOCs.

Processing Detector Data for Colorimetric Sensor Array

In embodiments utilizing a colorimetric sensor array based artificialnose, a detector 130 may be utilized to detect optical changes in thearray 120. In some embodiments, the detector 130 may only capture animage of the sensor array 120 before and at a single point in time afterexposure of the indicator 710 to the sample. In other embodiments, thedetector 130 may capture images at various times or continuously afterexposure of the sample to the indicator 710. The color changedifferences before and at various points after introduction of thesample are used to classify or determine the properties of the sample.For example, when used in combination with colorimetric sensor array 120and image analysis software, colorimetric differences can be generatedby subtracting the values of indicator images generated before and afterexposure of the indicator to a sample. In some embodiments, thecolorimetric differences represent hue and intensity profiles for thearray 120 in response to analytes contained in the sample. Thus, foreach image the detector 130 may extract a 240-dimensional 16-bit vector(R, G and B values if, for example, 80 indicators are used) before andafter exposure. When used in accordance with the method of the presentdisclosure, a unique color change signature for the sample can becreated which provides both qualitative recognition and quantitativeanalysis of volatile organic compounds present in the sample.

FIG. 4 illustrates an example of a process where certain or all of thesteps of may be implemented or controlled by a processing device 140,detector 130, associated database 160, server 150, and other electroniccomponents which communicate over a network. These computer or processorintegrated components can automatically implement the illustratedprocess to provide an indication of whether a patient has lung cancer.

For example, first, a sample may be exposed to an artificial nose 810.This may be implemented by a caregiver applying a sample inside acontainer 200 or a processing device 140 to open a door or other featureto allow exposure of the sample to the artificial nose 810 to begin.Exposure to the sample could be performed by any suitable method thatallows the headspace gas to be exposed to, come in contact with, or comewithin gaseous proximity to the colorimetric array 120. Next, a responseor image or several responses or images as described herein of thecolorimetric array or other artificial nose 820 may be captured by adetector 130. In some embodiments, the detector 130 will first capturean image or several images of the colorimetric array or artificial nose820 as a baseline prior to exposure of the colorimetric array 120 to thesample as described herein. Particularly, responses or readings fromtechnologies may be taken before and after introduction of the sample tothe artificial nose sensor or detector 130.

Next, the system may process the image data or other artificial noseresponse data 830 captured by the detector 130. The processing 830 maybe performed by any processing device 140 connected to the system. Forexample, the system may determine a colorimetric difference between thebaseline image and images captured after exposure of the colorimetricsensor array 120 to the headspace gas from the sample.

Then, the system may calculate or determine what matches the response.For instance, the system may calculate or determine a likelihood apatient from which the sample came has a malady such as cancer, aninfection from a microorganism, and certain information about thedisease or microorganisms including its susceptibility to antibioticsbased on the processing of the image or other detector 130 data. Inother examples, the system may determine the likelihood the samplematches a pollutant, a chemical warfare agent, or other chemical oranalyte.

For instance, this may be performed by comparing the colorimetricdifference determined in the processing 830 step to a database 160 tocolorimetric differences associated with samples belong to patients withknown ailments such as infections from microorganisms, cancer, or othermaladies. For example, a statistical analysis may be performed using anHCA or PCA analysis (as described more fully herein) to determine thelikelihood a sample indicates the associated patient has cancer, aninfection, or a drug resistant infection, based on comparisons todifferences in the samples in the database that are known to have canceror lung cancer or based on other processing techniques that filter andidentify certain features of the response.

Computer Implementation of Analysis

In order to implement the image based processing and analysis system,the system may also include a memory storage device operatively coupledto the processor that stores a multiplicity of temporal and/or staticcolor response patterns of known species and/or strains ofmicroorganisms (e.g., bacteria, yeast, protozoa). Thus, the system isoperable to generate a temporal and/or static color response pattern ofa sample, including a microorganism, and automatically identify themicroorganism (e.g., by species and strain) by comparing the generatedcolor response pattern of the array 120 with the stored multiplicity oftemporal and/or static color response patterns (e.g., the “library”) ofknown species and/or strains of microorganisms. Comparing the generatedcolor response pattern with the library of known species and/or strainsof microorganisms may be achieved by one of a number of statisticalmethods described herein or incorporated by reference.

In other embodiments, information output by detector 130 may be sent toa remote database to be processed and compared to a centralized databaseto determine the closest matching dataset. In other embodiments, certainportions of the calculation may be performed locally at a processor onthe system and some portions may be performed remotely by a processor orother computing device on a server. In some embodiments, a library ofdatasets with previous data points for known antibiotic strains and/orknown resistances or susceptibilities may be contained in the system orin a centralized server. In the server embodiments, the data could becontinually updated and stored as more assays are performed andorganisms identified along with susceptibilities.

The system may then output an indication of the likelihood 850 to adisplay associated with the system. The output of the indication may bepercentage likelihood a patient has cancer, an infection, a thresholddetermination of whether the patient should have follow up screening ortesting for cancer or infection, or further testing to validate theresults or other suitable indications. The systems and methods disclosedherein may also be able to provide additional quantitative informationregarding the diagnosis that may assist a patient in decision making.

Analyzing Images of Arrays

Analyzing the differences between the first image and the second imagemay include quantitative comparison of the digital images before andafter exposure to the analyte. Using customized software or standardgraphics software such as Adobe® PhotoShop®, a difference map can beobtained by subtracting the first image from the second image. To avoidsubtraction artifacts at the periphery of the spots, the center of eachspot can be averaged.

FIGS. 5A-5C are illustrations of an example image from a colorimetricsensor array, showing the array before exposure to E. coli ATCC 25922(FIG. 5A), after exposure to E. coli ATCC 25922 (FIG. 5B), and adifference map of these two images (FIG. 5C). The comparison dataobtained from the difference map includes changes in red, green and bluevalues (ARGB) for each spot in the array. The changes in spectralproperties that occur upon exposure to an analyte, and the resultantcolor difference map, can serve as a unique fingerprint for any analyteor mixture of analytes at a given concentration.

In the simplest case, an analyte can be represented by a single 3xvector representing the ΔRGB values for each colorant, where x is thenumber of colorants as set forth in equation (1). This assumes thatequilibration is relatively rapid and that any irreversible reactionsbetween analyte and colorant are slow relative to the initialequilibration time:

Difference vector=ΔR1,ΔG1,ΔB1,ΔR2,ΔG2,ΔB2, . . . ΔRx,ΔGx,ΔBx  (1)

Alternatively, the temporal response of the analyte can be used to makerapid identification, preferably using a “time-stack vector” of ΔRGBvalues as a function of time. In equation 2, a time-stack vector isshown for an array of 36 colorants at times m, n, and finally z, allusing the initial scan as the baseline for the differences in red, greenand blue values:

Time stack vector=ΔR1m,ΔG1m,ΔB1m,ΔR2m,ΔG2m,ΔB2m,−ΔR36m,ΔG36m,ΔB36m, . .. ΔRM,ΔG1n,ΔB1n, . . . ΔR36m,ΔG36m,ΔB36m, . . . ΔR36z,ΔG36z,ΔB36z   (2)

Accordingly, each analyte response can be represented digitally as avector of dimension 3xz, where x is the number of colorants and z is thenumber of scans at different times. Quantitative comparison of suchdifference vectors can be made simply by measuring the Euclideandistance in the 3xz space. Such vectors may then be treated by usingchemometric or statistical analyses, including principal componentanalysis (PCA), hierarchical cluster analysis (HCA) and lineardiscriminant analysis. Statistical methods suitable for highdimensionality data are preferred. As an example, HCA systematicallyexamines the distance between the vectors that represent each colorant,forming clusters on the basis of the multivariate distances between theanalyte responses in the multidimensional ΔRGB color space using theminimum variance (“Ward's”) method for classification. A dendrogram canthen be generated that shows the clustering of the data from theEuclidean distances between and among the analyte vectors, much like anancestral tree.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not intended to be interpreted as limiting the scopeof the invention. To the extent that specific materials or steps arementioned, it is merely for purposes of illustration and is not intendedto limit the invention. One skilled in the art may develop equivalentmeans or reactants without the exercise of inventive capacity andwithout departing from the scope of the invention.

Example 1: Detection of Microorganisms

Colorimetric sensor arrays described herein can be used to detect andidentify pathogenic and non-pathogenic microorganisms. In one example, asample including microorganisms from a mammal (e.g., a human) showingsymptoms of a malady or in need of treatment for a malady can be takenfrom the mammal (e.g., in the form of a fluid sample such as blood orexhaled breath, or in the form of a tissue sample) and cultured in thepresence of a colorimetric sensor array. In other examples,microorganisms such as Saccharomyces cerevisiae and others can bemonitored in processes such as baking and alcoholic fermentationprocesses, electricity generation in microbial fuel cells, and biofuelproduction.

Response of the sensors in the colorimetric sensor array to the volatileorganic compounds yields a strain-specific temporal or static colorresponse pattern, allowing the microorganism to be identified bycomparison of the color response pattern with color response patternsfor known strains. Comparison may be achieved, for example, visually orautomatically.

While bacteria of a given species share certain characteristics,different strains of the same species yield noticeably different colorresponse patterns (or “fingerprints”), allowing discrimination betweenstrains of the given species (e.g., between Staphylococcus aureus andmethicillin-resistant Staphylococcus aureus and between Enterococusfaecalis and vancomycin-resistant Enterococus faecalis). The colorresponse patterns allow identification of microorganisms by species andstrain and certain antibiotic resistant characteristics in a fraction ofthe time (e.g., about three-quarters of the time, about one-half of thetime, or about one-quarter of the time) of other methods, based at leastin part on conditions such as concentration, culture medium, cultureconditions (e.g., temperature), and the like.

Microorganisms such as bacteria, yeasts, protozoa, and fungi can beidentified as described herein. Species of bacteria that can beidentified include, for example, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus sciuri, Pseudomonas aeruginosa, Enterococcusfaecium, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae,Streptococcus pneumoniae, Streptococcus pyrogenes, Vibrio cholera,Achromobacter xylosoxidans, Burkholderia cepacia, Citrobacter diversus,Citrobacter freundii, Micrococcus leuteus, Proteus mirabilis, Proteusvulgaris, Staphylococcus lugdunegis, Salmonella typhi, StreptococcusGroup A, Streptococcus Group B, S. marcescens, Enterobacter cloacae,Bacillis anthracis, Bordetella pertussis, Clostridium sp., Clostridiumbotulinum, Clostridium tetani, Corynebacterium diphtheria, Moraxalla(Brauhamella) catarrhalis, Shigella spp., Haemophilus influenza,Stenotrophomonas maltophili, Pseudomonas perolens, Pseuomonas fragi,Bacteroides fragilis, Fusobacterium sp. Veillonella sp., Yersiniapestis, and Yersinia pseudotuberculosis. Strains of bacteria that can beidentified include, for example, S. aureus ATCC 25923, S. aureus ATCC29213, S. aureus ATCC 43300, S. aureus IS-13, S. aureus IS-38, S. aureusIS-43, S. aureus IS-70, S. aureus IS-120, S. aureus IS-123, S. aureusIS-124, methicillin-resistant S. aureus ATCC 33591, S. epidermidis ATCC35984, S. sciuri ATCC 49575, P. aeruginosa ATCC 10145, P. aeruginosaIS-15, P. aeruginosa IS-65, P. aeruginosa IS-22, P. aeruginosa IS-36, P.aeruginosa ATCC 27853, E. faecium ATCC 19434, E. faecalis ATCC 23241,vancoymcin-resistant E. faecalis ATCC 51299, E. coli ATCC 25922, E. coliATCC 53502, E. coli ATCC 35218, E. coli ATCC 760728, E. coli IS-39, E.coli IS-44, A. xylosoxidans IS-30, A. xylosoxidans IS-35, A.xylosoxidans IS-46, A. xylosoxidans IS-55, C. diversus IS-01, C.diversus IS-28, C. diversus IS-31, C. diversus IS-33, K. pneumoniaeIS-130, K. pneumoniae IS-133, K. pneumoniae IS-136, K. pneumoniae ATCC33495, B. anthrax Ames, B. anthrax UM23CL2, B. anthrax Vollum, Y. pestisC092, Y. pestis Java 9, S. epidermis 12228, S. epidermis IS-60, S.epidermis IS-61, P. miribilis IS-18, P. miribilis IS-19, P. miribilis12453, S. marcescens IS-48, S. marcescens IS-05, and S. marcescens13880, where “IS-#” refers to clinical isolates and the other strainsare ATCC® reference strains. Species of fungi that can be identifiedinclude, for example, Microsporum sp. Trichophyton sp. Epidermophytonsp., Sporothrix schenckii, Wangiella dermatitidis, Pseudallescheriaboydii, Madurella grisea, Histoplasma capsulatum, Blastomycesdermatitidis, Coccidioides immitis, Cryptococcus neoformans, Aspergillusfumigatus, Aspergillus niger, and Candida albicans. Similarly, yeastsincluding Ascomycota (Saccharomycotina, Taphyrinomycotina,Schizosaccharomycetes) and Basidiomycota (Agaricomycotina,Tremellomycetes, Pucciniomycotina, Microbotryomycetes) can be identifiedand, if desired, assessed for susceptibility. Examples includeSaccharomyces cerevisiae and Candida albicans. Protozoa includingflagellates (e.g., Giardia lamblia), amoeboids (e.g., Entamoebahistolytica), sporozoans (e.g., Plasmodium knowlesi), and ciliates(e.g., Balantidium coli) may also be identified as described herein.

Arrays with Nucleophilic Indicators for Detecting Microorganisms

Nucleic indicators 710 may be beneficial for detecting microorganisms ina closed environment such as a Petri dish or blood culture bottle, asmany for example many of the VOCs emitted by bacteria include ketones,aldehydes, and other molecules where concentrations of individualmetabolic VOCs range from 300 to 50,000 ppb.

Example 2: Detection of Cancer

Biomarkers for cancer from blood, saliva, and urine have beenidentified, including the following: proteins, tumor antigens,anti-tumor antibodies, cell type-specific peptides, metabolic productsand epigenetic phenomena such as hyper-methylated DNA, NRA, and theexpression of specific genes. However, to date, none of these biomarkershave had the adequate sensitivity, specificity, and reproducibility tobe utilized in an effective diagnostic test.

However, potential effective biomarkers for cancer may include lowmolecular weight volatile organic compounds, which can be detected inthe urine, breath and blood of patients. This evidence for cancermetabolism is expected to manifest as a characteristic concentrationprofile covering dozens of metabolic VOCs. For example, the volatileorganic compounds dimethyl succinate, 2-pentanone, phenol,2-methylpyrazine, 2-hexanone, 2-butanone and acetophenone, among others,have been found in increased concentrations in the urine headspace ofmice implanted with lung cancer cell lines. (Hani, et al., Analysis ofvolatile organic compounds released from human lung cancer cells andfrom the urine of tumor-bearing mice, Cancer Cell International, 2012,12:7).

Arrays with Nucleophilic Indicators for Detecting Colon Cancer

As discussed herein, many of the biomarkers for cancer cells may bedetected by nucleophilic indicators 710 as they are electrophilicanalytes. Accordingly, a study was performed to determine whetherbiomarkers for colon cancer could be detected in simulated breath. FIG.6 illustrates an example of a flow sensor 600 and sensor array 120 usedto detect VOCs in a breath of a patient. In this example, the sensorarray 120 included nucleophilic indicators 710. In this example, theflow sensor 600 is a linear quartz tube, and the sensor response ismeasured with a hyperspectral detector, that includes RGB color and fourUV bands.

In this example, simulated breath with different biomarkers was pumpedthrough the flow sensor 600. The simulated breath also contained 79% RH,16%02 and 5% C02. Selected examples of spiked biomarkers for coloncancer include N-methylphenylethanoamine, creatine, nonanal, decanal,4-methyl-2-pentanone, 4-methyloctane, m-xylene and p-xylene. Theresponses for each sensor 120 were compared before and after applicationof the breath. For the UV difference maps, sensor responses at threedifferent UV bands were used to create an artificial color map.

FIG. 7 illustrates those color and UV difference maps for the differentcolon cancer biomarkers and a control. Those color differences arefurther highlighted in FIG. 8, which illustrates two bar graphs showingsensor response (color change %) for the different biomarkers. Asillustrated, two of the nucleophilic indicators clearly indicated thepresence of electrophilic molecules that are reported biomarkers forcolon cancer. Additionally, the different responses for differentnucleophilic indicators for the same two biomarkers (nonanal & decanal)indicate the surprising result. Particularly, this demonstrates that anarray of more than one nucleophilic indicator could likely discriminateor identify which electrophilic analyte is present and potentiallyidentify patients with colon cancer by sensing their exhaled breath.

In some examples, this may allow the sensor array 120 and associatedsystem to determine which patients have cancer, and also what kind ofcancer. Accordingly, a sensor with a sensor array that includes severalnucleophilic indicators may be very powerful in identifying patientsamples that were extracted from patients with specific types ofcancers. For the aldehyde detection, o-dianisidine and2,3-diaminonaphthalene were most effective when used with a UVreflective optical system.

Example 3: Detection of Organophosphates

In another example, the disclosed arrays 120 with nucleophilicindicators were tested to see if they could identify organophosphates inliquid phase. FIG. 9 illustrates the array used that includes an array120 that is liquid phase based, so it relies on capillary action for theliquid to disperse to the outside of the star and contact the indicatorsor dyes 710.

FIG. 10 shows a difference map for the array 120. Those differences arefurther highlighted in FIG. 11, which illustrates bar graphs showing theresponse for four different nucleophilic indicators (indicator 8, 20,22, and 24) included in the array 120. The results demonstrated thatalthough indicator 8 is not effective against the six differentorganophosphates, the rest of the indicators show differential responseto the analytes over the solvent/control (isopropanol, IPA).

Accordingly, the example illustrates the potential for a nucleophilicarray to detect and potentially identify different types oforganophosphate analytes. As discussed, this may be important for thedetection of chemical warfare agents in the field, which could be testedwith liquid based sensor arrays.

Example 4: Detection of Formaldehyde

In another example, it was investigated whether sensor arrays withnucleophilic indicators could detect formaldehyde. FIG. 12 illustratestwo color differences maps. The first map represents the results of asensor printed on SG-81 (silica gel loaded cellulose paper) and testedagainst formaldehyde at different concentrations. The results show thecolor change after 10 minutes of exposure. The second color differencemap (bottom) is captured from a sensor printed on a polypropylenemembrane and tested against formaldehyde at the same concentrations.

FIG. 13 illustrates graphs of the color change response over time fortwo of the indicators on the SG-81 substrate 720. These graphsillustrate that the sensor can detect a response to formaldehyde even atthe 10 ppb level. FIG. 14 shows a graph that is based on the log of theconcentration. Accordingly, the array of nucleophilic indicators candetect formaldehyde at very low concentrations (10 ppb). With a safeexposure of 0.08 ppm, these sensors will be able to detect formaldehydewell before it becomes unsafe in the environment.

Additionally, the sensor response may be correlated to theconcentration, as illustrated in FIG. 14. Accordingly, the sensorresponse may also be useful for determining an airborne concentration offormaldehyde, and whether the environment is safe for humans.

CONCLUSIONS

Implementations of the subject matter and the operations described inthis specification can be implemented in digital electronic circuitry,or in computer software, firmware, or hardware, including the structuresdisclosed in this specification and their structural equivalents, or incombinations of one or more of them. Implementations of the subjectmatter described in this specification can be implemented as one or morecomputer programs, i.e., one or more modules of computer programinstructions, encoded on computer storage medium for execution by, or tocontrol the operation of, data processing apparatus. Alternatively, orin addition, the program instructions can be encoded on an artificiallygenerated propagated signal, e.g., a machine-generated electrical,optical, or electromagnetic signal that is generated to encodeinformation for transmission to suitable receiver apparatus forexecution by a data processing apparatus. A computer storage medium canbe, or be included in, a computer-readable storage device, acomputer-readable storage substrate, a random or serial access memoryarray or device, or a combination of one or more of them. Moreover,while a computer storage medium is not a propagated signal, a computerstorage medium can be a source or destination of computer programinstructions encoded in an artificially generated propagated signal. Thecomputer storage medium can also be, or be included in, one or moreseparate physical components or media (e.g., multiple CDs, disks, orother storage devices).

The data processing operations described in this specification can beimplemented as operations performed by a data processing apparatus ondata stored on one or more computer-readable storage devices or receivedfrom other sources.

The term “data processing apparatus” encompasses all kinds of apparatus,devices, and machines for processing data, including by way of example aprogrammable processor, a computer, a system on a chip, or multipleones, or combinations, of the foregoing. The apparatus can includespecial purpose logic circuitry, e.g., an FPGA (field programmable gatearray) or an ASIC (application specific integrated circuit). Theapparatus can also include, in addition to hardware, code that createsan execution environment for the computer program in question, e.g.,code that constitutes processor firmware, a protocol stack, a databasemanagement system, an operating system, a cross-platform runtimeenvironment, a virtual machine, or a combination of one or more of them.The apparatus and execution environment can realize various differentcomputing model infrastructures, such as web services, distributedcomputing and grid computing infrastructures.

A computer program (also known as a program, software, softwareapplication, script, or code) can be written in any form of programminglanguage, including compiled or interpreted languages, declarative orprocedural languages, and it can be deployed in any form, including as astand-alone program or as a module, component, subroutine, object, orother unit suitable for use in a computing environment. A computerprogram may, but need not, correspond to a file in a file system. Aprogram can be stored in a portion of a file that holds other programsor data (e.g., one or more scripts stored in a markup languagedocument), in a single file dedicated to the program in question, or inmultiple coordinated files (e.g., files that store one or more modules,sub programs, or portions of code). A computer program can be deployedto be executed on one computer or on multiple computers that are locatedat one site or distributed across multiple sites and interconnected by acommunication network.

The processes and logic flows described in this specification can beperformed by one or more programmable processors executing one or morecomputer programs to perform actions by operating on input data andgenerating output. The processes and logic flows can also be performedby, and apparatus can also be implemented as, special purpose logiccircuitry, e.g., an FPGA (field programmable gate array) or an ASIC(application specific integrated circuit).

Processors suitable for the execution of any computer programs disclosedherein include, by way of example, both general and special purposemicroprocessors, and any one or more processors of any kind of digitalcomputer. Generally, a processor will receive instructions and data froma read-only memory or a random access memory or both. The essentialelements of a computer are a processor for performing actions inaccordance with instructions and one or more memory devices for storinginstructions and data. Generally, a computer will also include, or beoperatively coupled to receive data from or transfer data to, or both,one or more mass storage devices for storing data, e.g., magnetic,magneto optical disks, or optical disks. However, a computer need nothave such devices. Moreover, a computer can be embedded in anotherdevice, e.g., a mobile telephone, a personal digital assistant (PDA), amobile audio or video player, a game console, a Global PositioningSystem (GPS) receiver, or a portable storage device (e.g., a universalserial bus (USB) flash drive), to name just a few. Devices suitable forstoring computer program instructions and data include all forms ofnon-volatile memory, media and memory devices, including, by way ofexample, semiconductor memory devices, e.g., EPROM, EEPROM, and flashmemory devices; magnetic disks, e.g., internal hard disks or removabledisks; magneto optical disks; and CD ROM and DVD-ROM disks. Theprocessor and the memory can be supplemented by, or incorporated in,special purpose logic circuitry.

To provide for interaction with a user, certain implementations and/orportions of the subject matter described in this specification can beimplemented on a computer having a display device, e.g., a CRT (cathoderay tube) or LCD (liquid crystal display) monitor, for displayinginformation to the user and a keyboard and a pointing device, e.g., amouse or a trackball, by which the user can provide input to thecomputer. Other kinds of devices can be used to provide for interactionwith a user as well; for example, feedback provided to the user can beany form of sensory feedback, e.g., visual feedback, auditory feedback,or tactile feedback; and input from the user can be received in anyform, including acoustic, speech, or tactile input. In addition, acomputer can interact with a user by sending documents to and receivingdocuments from a device that is used by the user; for example, bysending web pages to a web browser on a user's client device in responseto requests received from the web browser.

Implementations of certain portions of the subject matter described inthis specification can be implemented in a computing system thatincludes a back end component, e.g., as a data server, or that includesa middleware component, e.g., an application server, or that includes afront end component, e.g., a client computer having a graphical userinterface or a Web browser through which a user can interact with animplementation of the subject matter described in this specification, orany combination of one or more such back end, middleware, or front endcomponents. The components of the system can be interconnected by anyform or medium of digital data communication, e.g., a communicationnetwork. Examples of communication networks include a local area network(“LAN”) and a wide area network (“WAN”), an inter-network (e.g., theInternet), and peer-to-peer networks (e.g., ad hoc peer to-peernetworks).

Any computing systems disclosed herein can include clients and servers.A client and server are generally remote from each other and typicallyinteract through a communication network. The relationship of client andserver arises by virtue of computer programs running on the respectivecomputers and having a client-server relationship to each other. In someimplementations, a server transmits data (e.g., an HTML page) to aclient device (e.g., for purposes of displaying data to and receivinguser input from a user interacting with the client device). Datagenerated at the client device (e.g., a result of the user interaction)can be received from the client device at the server.

While this specification contains many specific implementation details,these should not be construed as limitations on the scope of anyinventions or of what may be claimed, but rather as descriptions offeatures specific to particular implementations of particularinventions. Certain features that are described in this specification inthe context of separate implementations can also be implemented incombination in a single implementation. Conversely, various featuresthat are described in the context of a single implementation can also beimplemented in multiple implementations separately or in any suitablesub-combination. Moreover, although features may be described above asacting in certain combinations and even initially claimed as such, oneor more features from a claimed combination can in some cases be excisedfrom the combination, and the claimed combination may be directed to asub-combination or variation of a sub-combination.

Similarly, while operations may be depicted in the drawings in aparticular order, this should not be understood as requiring that suchoperations be performed in the particular order shown or in sequentialorder, or that all illustrated operations be performed, to achievedesirable results. In certain circumstances, multitasking and parallelprocessing may be advantageous. Moreover, the separation of varioussystem components in the implementations described above should not beunderstood as requiring such separation in all implementations, and itshould be understood that the described program components and systemscan generally be integrated together in a single software product orpackaged into multiple software products.

Thus, particular implementations of the subject matter have beendescribed. Other implementations are within the scope of the followingclaims. In some cases, the actions recited in the claims can beperformed in a different order and still achieve desirable results. Inaddition, the processes depicted in the accompanying figures do notnecessarily require the particular order shown, or sequential order, toachieve desirable results.

1-18. (canceled)
 19. A method for determining if a patient has a malady,the method comprising: providing a sensor array comprising a firstindicator and a second indicator deposited on a substrate in apredetermined pattern wherein the first and second indicators arenucleophilic indicators; exposing the sensor array to a sample from apatient; detecting a spectral response after exposing the sensor arrayto the sample and correlating the spectral response to the presence of amalady.
 20. The method of claim 0 wherein the malady is cancer.
 21. Themethod of claim 0 wherein the malady is colon cancer.
 22. The method ofclaim 0 wherein the malady is sepsis.
 23. The method of claim 0 whereinthe sample is the exhaled breath of a patient.
 24. The method of claim 0wherein the sample is a urine sample.
 25. The method of claim 0 whereinexposing the sensor array to a sample from the patient comprisesexposing the sensor array to headspace gas of a blood sample.